The goal of the current proposal is to develop novel treatments for necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal disease in premature infants. NEC is thought to arise from an exuberant inflammatory response to bacterial colonization in the premature intestine through pathways that are incompletely understood. Based on studies in mice and humans, we previously discovered that the receptor for gram negative bacterial lipopolysaccharide, namely toll-like receptor 4 (TLR4), on the intestinal epithelium, is required for NEC development. TLR4 activity is higher in the premature human and mouse intestine compared to the full-term intestine, reflecting TLR4?s role in governing gut development. The subsequent activation of TLR4 by colonizing bacteria leads to inflammation and NEC. In seeking to extend these studies, we will now explore the upstream pathways that restrain TLR4 signaling, and which become untethered leading to NEC. We have determined that the enteric glia in the premature gut inhibit intestinal TLR4 through the release of brain derived neurotrophic factor (BDNF), while the relative lack of enteric glia in the premature bowel predisposes to NEC through exaggerated TLR4 signaling. In support of this observation, we generated three strains of glial-deficient mice that show elevated newborn intestinal epithelial TLR4 signaling and increased NEC severity, which were reversed by BDNF, while mice and humans with NEC show reduced enteric glia and BDNF, leading to increased TLR4 signaling. Enteric glia loss depended on TLR4 activation on the enteric glia themselves, since mice lacking TLR4 on enteric glia were protected from glial loss and NEC, suggesting that strategies which promote enteric glial survival could treat NEC. Strikingly, a library screen yielded a new class of molecules, of which our lead is ?J11?, which enhanced enteric glia BDNF release in both the premature mouse and human intestine ex vivo, and prevents experimental NEC. Based upon the above preliminary data, we hypothesize that the enteric glial play a critical, but previously unrecognized, role in restraining the exaggerated signaling of TLR4 in the intestinal epithelium thus preventing NEC, and that this protective effect occurs through the release of BDNF. We further hypothesize that the paucity of enteric glia in the premature gut, in combination with TLR4-induced enteric glial loss, leads to exaggerated epithelial TLR4 signaling and NEC. Finally, we hypothesize that our recently discovered enteric glial activating agent, ?J11?, may represent a novel NEC prevention and treatment strategy. We will test these hypotheses in 3 aims: Aim 1. Determine mechanisms by which the enteric glia restrain TLR4 signaling in the intestinal epithelium in the pathogenesis of NEC; Aim 2. Determine mechanisms of enteric glia loss in NEC, and augment enteric glia effects to prevent NEC; Aim 3. Evaluate ?J11? for the prevention and treatment of NEC in mice and piglets. These studies promise to offer novel therapeutic approaches for children with NEC, based upon enteric glial effects on TLR4 within the premature intestine.